The question of how the epidermal appendages form during development and which pathways regulate the differentiation of the stratified epidermis has been central to epithelial biology. Transcription is one of the most important regulatory mechanisms controlling the stepwise program of epidermal differentiation. Epidermis has been used as an excellent model for studying the process of cellular differentiation because the cells form a stratified structure during development, and each stratum is easily identified by morphology and expression of specific markers. Our research effort have focused in characterizing the regulation and function of Dlx3 homeobox transcription factor, a member of the murine Dlx family, with essential roles in epidermal, osteogenic and placental development.[unreadable] Dlx homeodomain proteins are a family of transcription factors that play crucial roles in developmental processes and differentiation of individual tissues. The Dlx3 gene is broadly expressed in the branchial arches and embryonic ectoderm, as well as in the tooth, hair follicle and interfollicular epidermis. To elucidate the functional role of the Dlx3 homeodomain protein during early development and during epidermal differentiation, we are currently in the process of determining the target genes and the Dlx3 interacting factors necessary to exert the transcription regulatory function. For the comprehensive analysis of Dlx3 gene expression throughout development, we generated a knock-in mouse carrying the reporter gene beta-galactosidase (LacZ) under the control of the endogenous Dlx3 promoter. Whole-mount or local X-gal staining shows the temporal and spatial expression of the Dlx3 gene in various tissues and organs including hair, teeth and bone. Targeted deletion of Dlx3 results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). We are currently in the process of completing the analysis of the epidermal-conditional ablation of Dlx3 to be able to specifically assess the role of this homeodomain transcription factor in epidermal development. Recent work also showed that Dlx3 induction coincides with the onset of commitment to osteogenic lineage, as reflected by the induction of bone-related differentiation genes. Although Dlx3 had been implicated in skeletal development in the human autosomal dominant Tricho-Dento-Osseous (TDO) syndrome, where the mutation is associated with defects in the bone, a direct role for Dlx3 in bone formation had not been identified. In a collaborative effort with Dr. Lian's laboratory, a regulatory role for Dlx3 in bone-specific transcriptional control during osteoblast development was demonstrated.[unreadable] During embryonic development and organ formation, a series of signals between epithelial cells and underlying mesenchymal cells, leads to the formation of a variety of appendages/organs. Anomalies in epithelial/mesenchymal-derived organs are characteristics of a group of about 150 human heritable pathological disorders defined as ectodermal dysplasias (EDs). DLX3 is among the few genes for which mutations have been directly linked with EDs. The importance of Dlx3 in the patterning and development of ectodermal structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of DLX3 mutations in patients with the autosomal dominant TDO syndrome. Recent research on the mechanistic role of Dlx3 in epidermal development and appendage formation has led to the important finding that Dlx3 is a direct target of the p63. This work presents the crucial finding that besides autosomal mutations in either DLX3 or P63 genes being associated with human EDs, misregulation of DLX3 function is directly involved in the pathogenesis of ectodermal human syndromes associated to p63 molecular lesions.[unreadable] We are also continuing with the characterization of a novel Ca++-binding protein identified in the laboratory. The Ca++ signaling dependent systems, such as keratinocyte differentiation process, must be finely tuned for rapid and effective response to transient variations in Ca++ concentration. A central role in the transduction of Ca++ signals is played by members of the Ca++-binding proteins. The functionality of the EF-hand motifs may contribute to specificity in the interaction with the target molecules. Thus, it is through the binding of Ca++ by the EF-motifs that these proteins are able to bind or liberate its target interacting proteins and in this way modulate their function. Performing affinity columns and subsequent mass spectrometry analysis, we have identified several Scarf target proteins. We are finalizing the characterization of the targeted deletion of Scarf during embryonic development.